[CANCER RESEARCH 61, 6868 – 6875, September 15, 2001] Antigenicity of Fusion Proteins from Sarcoma-associated Chromosomal Translocations 1 B. Scott Worley, Leon T. van den Broeke, Theresa J. Goletz, C. David Pendleton, Emily M. Daschbach, Elaine K. Thomas, Francesco M. Marincola, Lee J. Helman, and Jay A. Berzofsky 2 Metabolism Branch [B. S. W., L. T. v. d. B., T. J. G., C. D. P., E. M. D., J. A. B.] and Pediatrics Branch [L. J. H.], National Cancer Institute, Bethesda, Maryland 20892; Immunex Corporation, Seattle, Washington 98101-2936 [E. K. T.]; and Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, Maryland 20892 [F. M. M.] ABSTRACT Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5 region of one gene with the 3 region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Spe- cific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunolog- ical relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously express- ing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the develop- ment of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations. INTRODUCTION Neoplastic transformation occurs, in part, from the inactivation of tumor suppressor genes and/or the activation of proto-oncogenes. In some cases, the latter occurs as a result of chromosomal abnormalities such as translocations or inversions. Mechanistic theories of chromo- somal translocations have been reviewed extensively (1–5). One mechanism involves a break within the coding sequence of each constituent gene, which generates a functional chimeric gene. This occurs primarily in sarcomas and leukemias, and the affected genes often encode transcription factors, suggesting a role in the transfor- mation process. The fusion product retains the DNA binding speci- ficity of one gene while inappropriately activating or repressing transcription through the transactivation domain of the other gene. This phenomenon is exclusive to the tumor cell and thus provides a tumor-specific marker. Among solid tumors, SS, 3 CCS, and DSRCT are connective tissue-related malignancies that primarily affect adolescents and young adults. SS is an aggressive malignancy that occurs predom- inantly in the extremities and accounts for 5–10% of soft-tissue sarcomas. PCR analysis has shown that over 90% of SS cases contain a characteristic t(X;18)(p11.2;q11.2) translocation (6), which fuses the NH 2 -terminal region of SYT with the COOH- terminal region of SSX1 or SSX2 (6 – 8). In the chimeric fusion protein, a repression domain from SSX is replaced by a transacti- vation domain from SYT, and this presumably activates unknown target genes normally repressed by SSX1 or SSX2 (9). CSS, also termed malignant melanoma of soft parts, is a rare, aggressive sarcoma of neuroectodermal origin (10) affecting muscle tendons and aponeuroses typically in the extremities (11). Over 70% of these tumors contain the t(12;22)(q13;q12) translocation, fusing the 5' region of EWS with the 3' region of ATF1 (12, 13). Whereas it is not precisely known how the translocation contributes to cellular transformation, a number of mechanisms have been postulated (3), including constitutive activation of ATF1 target genes, repression of growth control genes, and activation of other cAMP-responsive ele- ment binding protein (CREB)/ATF or non-CREB/ATF target genes. DSRCT is an aggressive malignancy occurring predominantly in abdominal serosal surfaces (13). Almost 100% of these cases contain a characteristic t(11;22)(p13;q12) translocation that fuses the NH 2 - terminal region of EWS with the COOH-terminal region of WT1,a tumor suppressor gene involved in a subset of Wilms’ tumors (14 – 18). WT1 contains three zinc fingers in the COOH-terminal region responsible for DNA binding, and it has been postulated that the loss of the proximal zinc fingers in the chimeric fusion protein converts WT1 from a transcriptional repressor to an activator (3, 19). Resulting abnormalities in WT1 target gene expression, including genes related to the early growth response family, could be causative in cellular transformation (3). It has been shown that chimeric fusion proteins resulting from translocations can be necessary for the persistence of the tumor (20, 21), thus indelibly marking the tumor cell as a target for immunotherapy. Peptides generated from proteolytic processing of fusion regions would have a “non-self” sequence of aa, which, if displayed via MHC molecules, could result in T-cell-mediated immu- nity. Normal cells, on the other hand, would not contain the break- point-spanning sequence, thus distinguishing specific tumor cells and avoiding an autoimmune response. In the case of alveolar rhabdomyo- sarcoma, a common pediatric soft-tissue sarcoma caused by fusion of the NH 2 -terminal region of PAX3 with the COOH-terminal region of FKHR (22–26), it has previously been found that a PAX3-FKHR- induced CTL line lysed murine tumor cells transfected with the full-length PAX3-FKHR cDNA (27). This demonstrates that fusion proteins can be endogenously processed in tumor cells and presented by class I MHCs on the cell surface. In the following study, peptides derived from the sequences sur- rounding the breakpoints of SS, CCS, and DSRCT were assessed for the ability to bind HLA molecules and induce a primary in vitro Received 5/1/01; accepted 7/18/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the G. Harold and Leila Y. Mathers Foundation. 2 To whom requests for reprints should be addressed, at Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, NIH, Building 10, Room 6B-12 (MSC#1578), 10 Center Drive, Bethesda, MD 20892-1578. Phone: (301) 496-6874; Fax: (301) 496-9956; E-mail: berzofsk@helix.nih.gov. 3 The abbreviations used are: SS, synovial sarcoma; CCS, clear cell sarcoma; DSRCT, desmoplastic small round cell tumor; ATF, activating transcription factor; FBS, fetal bovine serum; ATCC, American Type Culture Collection;  2 m,  2 -microglobulin; FI, fluorescence index; IL, interleukin; aa, amino acid(s). 6868 on April 2, 2016. © 2001 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from